Odor perception in mammals is a complex process mediated by the activation of a family of G- protein coupled receptors (GPCRs) known as odorant receptors (ORs) expressed at the cilia of millions of olfactory sensory neurons (OSNs) lining the olfactory epithelium. The olfactory epithelium is richly innervated by autonomic nerves, including parasympathetic nerve endings which release acetylcholine. We recently discovered that OSNs express the type 3 muscarinic acetylcholine receptor (M3R), another GPCR. However, the molecular mechanisms underlying cholinergic actions on OSNs are not well-understood. We propose experiments to address the importance of modulation of olfaction and to clarify the molecular mechanisms that underlie the effects of acetylcholine receptors on OSN signaling. This work will further our understanding of how information processing occurs during the earliest steps of olfaction. To test the hypothesis that the M3R is important for the regulatio of OR function and olfactory signal transduction, the following Specific Aims are proposed: Aim 1. To test the hypothesis that M3R modulates the function of olfactory sensory neurons Aim 2. To test the hypothesis that M3R is involved in odor-mediated activation of alternative signaling Aim 3. To identify structural domains of M3R and signaling components important for the functional interaction of M3R and OR The proposed experiments further our long term goal, which is to understand the mechanistic basis for regulation of odorant receptor function. The health relatedness of these studies is that a better understanding of the regulatory mechanism of ORs and downstream signal transduction will offer insights into the general features of signaling by GPCRs, the most frequent pharmaceutical targets for cardiac, psychiatric, and cancer diseases. This work will advance our understanding on how G protein-coupled ORs are regulated by another GPCR, M3R, which is activated by acetylcholine, the key neurotransmitter of the parasympathetic nervous system. An understanding of the interactions between the neurotransmitter receptor and the peripheral olfactory system provides crucial information that may contribute toward novel therapy for anosmic and hyposmic patients who have compromised sense of smell, and to reduce stress associated with malodor, improving their quality of life. PUBLIC HEALTH RELEVANCE: The health relatedness of these studies is that a better understanding of the regulatory mechanism of ORs and downstream signal transduction will offer insights into the general features of signaling by GPCRs, the most frequent pharmaceutical targets for cardiac, psychiatric, and cancer diseases. This work will advance our understanding on how G protein- coupled ORs are regulated by another GPCR, M3-R, which is activated by acetylcholine, the key neurotransmitter of the parasympathetic nervous system. An understanding of the interactions between the neurotransmitter receptor and the peripheral olfactory system provides crucial information that may contribute toward novel therapy for anosmic and hyposmic patients who have compromised sense of smell, and to reduce stress associated with malodor, improving their quality of life.